Plants and seeds provide an important source of dietary protein for humans and livestock. However, the protein content of plants and seeds is often incomplete. For example, many plant and seed proteins are deficient in one or more essential amino acids. This deficiency may be overcome by genetically enhancing the native proteins to have a more nutritionally complete composition of amino acids (or some other desirable feature). Alternatively, a non-native (or heterologous) protein exhibiting a desirable characteristic may be introduced into the plant or seed. These approaches are useful in producing proteins exhibiting important agricultural (e.g., insecticidal), nutritional, and pharmaceutical properties.
Despite the availability of many molecular tools, the genetic modification of plants and seeds is often constrained by an insufficient accumulation of the engineered protein. Many intracellular processes may impact the overall protein accumulation, including transcription, translation, protein assembly and folding, methylation, phosphorylation, transport, and proteolysis. Intervention in one or more of these processes can increase the amount of protein produced in genetically engineered plants and seeds.
For example, raising the steady-state level of mRNA in the cytosol often yields an increased accumulation of translated protein. Many factors may contribute to increasing the steady-state level of an mRNA in the cytosol, including the rate of transcription controlled by promoter strength and other regulatory features, efficiency of mRNA processing, and the overall stability of the mRNA.
Among these factors, the promoter portion of a gene plays a central role. Along the promoter region, the transcription machinery is assembled and transcription is initiated. This early step is often rate-limiting relative to subsequent stages of protein production. Transcription initiation at the promoter may be regulated in several ways. For example, a promoter may be induced by the presence of a particular compound, express a gene only in a specific tissue, or constitutively express a coding sequence. Thus, transcription of a coding sequence may be modified by operably linking the coding sequence to promoters with different regulatory characteristics.
The promoters derived from the genes of seed storage proteins often exhibit high levels of expression. For example, seeds of Phaseolus vulgaris typically contain large amounts of phaseolin (36-46%, w/w), globulin-2 (5-12%), albumin (12-16%), and prolamine (2-4%). Thus, the promoters derived from such genes may be useful in expressing high levels of heterologous structural nucleic acid sequences.
However, the transcriptional activity of even these strong promoters may vary from one plant context to the next. For example, a number of promoters have exhibited strong activity in tobacco, petunia, and Arabidopsis, relative to the expression levels generated in these plants by the 7Sα′ promoter. However, none of these promoters have been reported to demonstrate comparable activity in transgenic soybean plants. Thus, a promoter which functions in one plant species or cultivar may not function at a similar level or manner in a different plant species or cultivar.
Romero et al. reported a new seed protein in P. vulgaris collected at Arcelia, Mexico (from accession PI 325690; CIAT No. 12882B). Accordingly, the protein was named Arcelin (Andreas et al., 1986; Osborn et al., 1986). Several Arcelin variants have been subsequently reported (e.g., Arcelin-3 from accession PI 417683 (CIAT No. G12922); Arcelin-4 from accession PI 417775 (CIAT No. G12949)). One such variant, designated Arcelin-5, was reported by Lioi, et al (Lioi and Bollini, 1989). The cDNA of Arcelin-5 was described by Goossens, et al. (Goossens et al., 1994).
A genomic clone of Arcelin-5, including an undefined 5′ and 3′ region was reportedly expressed in transgenic plants. This undefined region included about 1.8 kb base pairs 5′ to the Arcelin-5 coding region. Expression was reported in Arabidopsis and Phaseolus acutifolius (Goossens et al., 1999). However, the expression was lower than that found in the wild-type P. vulgaris from which Arcelin-5 was originally identified. Therefore, the genetic background is important in modulating Arcelin-5 expression. Also, since the whole genomic clone of Arcelin-5 was used by Goosens, the relative strength of the Arcelin-5 promoter was not clear. Thus although expression of an Arcelin species was reported, the effectiveness of such Arcelin promoters in crops such as maize and soybean remain totally unknown. Consequently, there is a need in the art for promoters capable of generating relatively high levels of transcription in important crop species, such as maize and soybean.